Project Summary/Abstract. The inability to maintain abstinence is a trademark of addiction yet effective maintenance therapies remain elusive. Women may face unique issues with substance abuse treatment, as research indicates that psychological and biological responses to drugs of abuse differ in women compared to men. Women tend to show greater drug dependence, progress more quickly from casual drug use to dependence, have greater difficulty quitting, and have shorter periods of abstinence than men. These effects have been similarly observed in female animal models. Thus, there is a crucial need for understanding circuit dynamics and molecular signatures that drive robust behavioral sex differences impeding abstinence success. We have developed a novel behavioral model termed the seeking-persistence paradigm (SPP) that was adapted from standard drug abstinence paradigms. The seeking-persistence paradigm is used to investigate the influence of intervention during initial abstinence on long-term drug-seeking behaviors. Indeed, cocaine-seeking during initial abstinence strongly correlates with seeking after drug-abstinence. These results reflect clinical studies indicating that craving during initial abstinence predicts relapse rates. Using this paradigm in combination with electrophysiological, chemogenetic, and functional genomic methods, we aim to ultimately provide novel targets for therapeutics by investigating the role of sex-specific signaling in driving cocaine seeking persistence. We have previously identified that the dorsal hippocampus CA1 is a crucial downstream target for noradrenergic (NE) modulation of cocaine-seeking on ED1. Notably, the locus coeruleus (LC) (NE)-CA1 signal in this behavior appears female- specific, and we hypothesize that recruitment of this pathway engages increased cocaine-seeking observed in females. In year 1 of the K99 phase, I will first determine if this sex difference involves phasic or tonic locus coeruleus firing on ED1 using unit recordings in behaving rats. In year 2 of the K99 phase, I will use RNA- sequencing and bioinformatics analyses to identify a molecular signature in CA1 of increased cocaine-seeking behavior. Next, in my independent phase (R00) I will combine prior training in chemogenetic techniques with new training in awake behaving electrophysiology to identify the effects of inhibition or activation of LC inputs to dorsal hippocampus CA1 on ED1 cocaine seeking and cocaine seeking-persistence, using PRSx8 promoter driven designer receptors exclusively activated by designer drugs (DREADDs) that permit specific expression in LC-NE neurons. Subsequently, I will use my bioinformatics training to identify clusters of transcripts that associate with cocaine-seeking resilience by examining transcripts that negatively correlate to cocaine-seeking behavior. These experiments will therefore characterize the role of the LC(NE)-CA1 pathway in driving cocaine- seeking behavior and identify novel gene targets in CA1 associated with sex differences in cocaine-seeking. The ultimate goal of this research is to identify the behavioral significance of neural activity (Aim 1), activated transcripts (Aim 2), and recruited circuits (Aim 3) driving sex differences in cocaine-seeking SPP.